A Research Protocol to Study the Dose- and Age-Dependent Effects of Breathing Low Oxygen on Enhancing Transcutaneous Spinal Stimulation-Induced Walking Function for People with Spinal Cord Injury: The BO 2 ST-II Trial

Abstract

Improving overground walking in people with chronic, incomplete spinal cord injury (iSCI) is especially important for older adults, who now represent more than 40% of the SCI population. Age and injury appear to interact to limit neuroplasticity in spared pathways, constraining gains from gait training. Brief episodes of low oxygen breathing (therapeutic acute intermittent hypoxia [tAIH]) may function as a plasticity-promoting primer to enhance the effects of transcutaneous spinal stimulation-augmented gait training (WALK_tSTIM), particularly in older individuals. This multicenter, age-stratified, placebo-controlled clinical trial will examine dose- and age-dependent effects of tAIH+WALK_tSTIM on walking recovery in 60 adults with chronic (>1 year) iSCI. After an eight-session WALK prewash phase, participants are randomized within age strata (18-49 and 50-80 years) to tAIH+WALK_tSTIM or Placebo+WALK_tSTIM. The active intervention delivers daily tAIH or Placebo immediately before a 45-min WALK_tSTIM session, 4 days per week for 4 weeks. Primary outcomes are walking speed (10-Meter Walk Test), endurance (6-Minute Walk Test), and balance (Timed Up-and-Go), assessed at baseline, during intervention weeks, and 1, 4, and 8 weeks post intervention. Safety and mechanistic measures include lower extremity strength, spasticity, pain, cardiopulmonary function, cognition, autonomic events, and serum testosterone. Findings will determine whether tAIH priming augments WALK_tSTIM relative to Placebo, whether extending pretreatment from 2 to 4 weeks (in pooled analyses with BO₂ST-I) yields larger or more durable walking gains, and whether age and hormone levels modify responsiveness. These results will inform age-appropriate tAIH dosing strategies and support the development of personalized neuromodulatory rehabilitation for an aging SCI population.

Keywords

aging electrical stimulation hypoxia neuronal plasticity neurological rehabilitation spinal cord trauma walking

Introduction

Spinal cord injury (SCI) disrupts communication between the brain, spinal cord, and body, often causing sensorimotor deficits that lead to lifelong paralysis. Most injuries are incomplete (incomplete SCI [iSCI]),¹ leaving spared neural pathways that can support functional recovery, particularly in walking, which is consistently ranked as a top recovery priority by people with chronic iSCI.² Walking performance, especially speed, also serves as a practical marker of overall health and independence,³ yet spontaneous gains in walking typically plateau by 12 months post-injury, underscoring the need for targeted interventions in the chronic phase.⁴

Emerging neuromodulation strategies that harness intrinsic neuroplasticity offer promising avenues to enhance walking recovery after iSCI. Therapeutic acute intermittent hypoxia (tAIH) induces serotonin- and brain-derived neurotrophic factor (BDNF)-dependent plasticity,⁵ preserves premotor and motor neurons in pre-clinical models,⁶ and can improve lower limb strength and overground walking when paired with gait training. Transcutaneous spinal stimulation (tSTIM) increases the excitability of lumbosacral networks by activating spared afferent and descending pathways,⁷ thereby amplifying the effects of skill-based gait training on stepping and standing. Clinical trials involving individuals with chronic iSCI indicate that even a single session of tAIH can improve lower limb strength⁸; furthermore, daily application of tAIH over several days facilitates improvements in overground walking ability, particularly when used as a priming technique prior to skill-based walking training (tAIH + WALK).^9–11 Recent research by McKenzie et al. corroborates these findings, demonstrating that tAIH pretreatment to WALK_tSTIM yielded greater gains in walking speed than WALK alone or with tSTIM in a small cohort of persons with chronic iSCI.¹² Although the exact neural mechanisms of tSTIM-related sensorimotor gains are not fully understood, evidence suggests that tSTIM activates spared afferent pathways and descending inputs, depolarizing spinal interneurons and motor neurons⁷ and thereby enhancing the effects of skill-based training on affected limbs.

Treatment responsiveness, however, is highly variable, and age-related factors appear to play a key role. Many studies show that individuals with traumatic SCI who are over 50 years old are less likely to regain functional walking than younger individuals.^13–15 Accumulating evidence links this disparity to age- and injury-related declines in sex hormones, particularly testosterone and its conversion to estradiol.¹⁶

These hormones modulate BDNF expression and serotonin receptor availability,¹⁷ both of which are central to tAIH-induced plasticity, suggesting that hormonal changes with aging and SCI may blunt responsiveness to tAIH priming. When basal testosterone levels are low, its plasticity-related benefits may depend primarily on its conversion to estradiol and on estrogen receptor activation, which regulate BDNF signaling and serotonin receptor availability.^18,19 However, the extent to which these hormonal shifts limit walking-related plasticity in older adults with iSCI has not been systematically tested.

The BO₂ST-II Trial is designed to address this gap by using an age-stratified, double-blind, placebo-controlled design to examine how age and hormone status influence responsiveness to tAIH delivered before WALK_tSTIM in adults with chronic iSCI. Younger (18–49 years) and older (50–80 years) cohorts will be studied to determine whether age modifies the magnitude, time course, and durability of tAIH-primed walking gains and to identify age-appropriate tAIH parameters. In parallel, this trial will assess how treatment dose, operationalized as the number of tAIH sessions, contributes to long-term improvements in overground walking. Together with the preceding BO₂ST-I Trial,²⁰ which uses a 2-week tAIH pretreatment, BO₂ST-II extends tAIH exposure to 4 weeks, enabling pooled analyses to define the dose-response profile of tAIH pretreatment and its capacity to optimize WALK_tSTIM for long-term walking recovery in an aging iSCI population.

Hypotheses

This study tests the overarching hypothesis that tAIH pretreatment before WALK_tSTIM safely boosts training-related improvements in overground walking in adults with chronic iSCI. Using a well-established tAIH protocol known to enhance walking in this population, we will assess whether adding more tAIH-primed WALK_tSTIM sessions results in larger and more lasting gains in walking ability. Hypothesis 1 (efficacy and durability):

Daily tAIH+WALK_tSTIM (vs. Placebo+WALK_tSTIM) will improve overground walking speed and endurance at 4 weeks (16 sessions) compared with 2 weeks (8 sessions), and these improvements will persist 8 weeks after treatment.

Hypothesis 2 (age effects):

Daily tAIH+WALK_tSTIM will improve overground walking recovery related to training more in younger than in older adults with chronic iSCI.

Hypothesis 3 (safety):

Daily tAIH+WALK_tSTIM will not increase maladaptive outcomes compared with Placebo+WALK_tSTIM.

By testing these hypotheses, this trial will clarify the functional benefits of tAIH priming, determine how age and dose (number of sessions) influence responsiveness, and guide the development of age- and dose-specific neuromodulatory strategies that can be integrated into best practices for SCI rehabilitation.

Methods

Study design

This is a double-blinded, placebo-controlled, age-stratified, randomized clinical trial to examine the age- and dose-related effects of tAIH+WALK_tSTIM on overground walking recovery in adults with chronic iSCI. The trial is taking place at two clinical sites: Spaulding Rehabilitation Hospital (Boston/Cambridge, MA) and Brooks Rehabilitation (Jacksonville, FL). The study received approval from the Mass General Brigham Institutional Review Board (MGB IRB; #2024P001608) and the Department of Defense Office of Human Research Oversight in April 2025. Any protocol amendments will be approved by the IRB before implementation. Enrollment in the study began in August 2025.

Sample size

We plan to enroll 60 adults with chronic iSCI (Fig. 1), which provides sufficient power for the primary efficacy analyses. This target sample size is based on conservative estimates from a previous placebo-controlled trial where individuals with iSCI improved their 10-Meter Walk Test (10MWT) time after tAIH + WALK compared with Placebo + WALK, with an estimated standard deviation of 2.0 sec for the change in 10MWT time in both groups.⁹ Assuming a 20% dropout rate, three primary walking measures (10MWT, Timed Up-and-Go [TUG], and 6-Minute Walk Test [6MWT]), and a Bonferroni-adjusted significance level of 0.0167 with the overall type I error rate of 5%, this sample size offers high power to detect differences between groups in walking performance.

FIG. 1.

Flow diagram of anticipated subject recruitment, enrollment, treatment allocation, and disposition status. To prevent the potential of early dropouts causing an imbalance in treatment randomization, group assignment will occur immediately prior to the treatment phase. We anticipate that 60 people with SCI will participate in the study. SCI, spinal cord injury.

We also examined data from a small preliminary crossover study (unpublished) in which three participants with chronic iSCI showed improved walking speed after five consecutive days of tAIH+WALK_tSTIM, with two reaching a minimal clinically important difference (MCID) on the 10MWT. In contrast, no participants reached an MCID after 5 days of Placebo+WALK_tSTIM. In that pilot, we estimated the standard deviation of the number of sessions needed to reach the MCID at about 1.5 sessions, supporting over 95% power to detect a difference between 5 sessions (tAIH+WALK_tSTIM) and 7 sessions (Placebo+WALK_tSTIM) with 48 completers (accounting for 20% dropout).

Randomization

Participants will be divided into age groups (younger: 18–49 years; older: 50–80 years) and then randomly assigned to receive tAIH+WALK_tSTIM or Placebo+WALK_tSTIM. Within each age group, participants will be randomly assigned in a 2:1 ratio to tAIH+WALK_tSTIM or Placebo+WALK_tSTIM using permuted blocks of size 6, aiming for a total of 60 participants (30 in each age group). Sex will not be included as a stratification variable, as enrollment is expected to reflect the predominantly male demographic of the SCI population.¹ The study biostatistician generated the randomization scheme prior to enrollment and will implement it separately at each site. Allocation is concealed from participants and the study team members involved in interventions and assessments.

Study recruitment and enrollment

We are recruiting participants for this study at two sites: Spaulding Rehabilitation (Boston, MA) and Brooks Rehabilitation (Jacksonville, FL). Our study recruitment resources include site-specific patient registries, clinicaltrials.gov, IRB-approved flyers, and clinician referrals. Potential participants undergo screening to determine eligibility (Table 1), which includes a medical records review and clearance from a physician. As part of this screening, we confirm the following: American Spinal Injury Association Impairment Scale (AIS) grade C or D, ability to walk 10 m without human assistance, a score of ≥25 on the Mini-Mental State Examination (MMSE), and that their lower extremity involuntary motor threshold is within the range of the stimulator (≤200 mA). Additionally, we screen for sleep-disordered breathing using a portable sleep monitor (Nox A1s, Nox Medical, Suwanee, GA; ApneaLink, ResMed Inc., San Diego, CA; or WatchPAT, ZOLL Itamar Medical, Atlanta, GA) at home for one night.

Table 1.

Enrollment Criteria

Inclusion criteria
• 18–80 years of age
• Medically stable with medical clearance from study physician to participate
• iSCI at and between levels C1 and L2 with at least some sensory or motor function preserved below the neurological level to ensure phrenic and cauda equina sparing
• Non-progressive etiology of iSCI
• AIS²¹ Grades C–D
• Ambulatory, able to walk 10 m without support from another person
• Chronic iSCI (≥12 months post-injury)^a
Exclusion criteria
• Severe concurrent illness or pain^b
• Cognitive impairment measured by a score of <24 on the MMSE²²
• Severe recurrent autonomic dysreflexia per assessment by the study physicians
• History of severe cardiovascular/pulmonary complications including hypertension (systolic blood pressure >150 mmHg)
• Pregnancy^c
• Botulinum toxin injections in lower extremity muscles within the last 3 months²³
• History of tendon or nerve transfer surgery in the lower extremity
• Untreated severe sleep-disordered breathing^d
• Active implanted devices (e.g., intrathecal baclofen pump)
• Receiving concurrent electrical stimulation
• tSTIM-induced involuntary motor threshold is >200 mA (exceeding SCONE’s current limit)²⁴

Chronic iSCI to avoid the potential confounding effects of spontaneous plasticity.^25,26

Illness includes unhealed decubiti, severe neuropathic or chronic pain syndrome, severe infection (e.g., urinary tract), hypertension, cardiovascular disease, pulmonary disease, severe osteoporosis, active heterotopic ossification in the lower extremities, and severe systemic inflammation.

Pregnancy is an exclusion criterion due to unknown effects of tAIH or tSTIM on a fetus; individuals of childbearing potential will not otherwise be excluded.

Severe sleep-disordered breathing >30 apneas or hypopneas per hour.²⁷

iSCI, incomplete spinal cord injury; AIS, American Spinal Injury Association Impairment Scale; MMSE, Mini-Mental State Examination; tAIH, therapeutic acute intermittent hypoxia.

Potential study participants who meet eligibility criteria must sign IRB-approved consent forms during an informed consent process prior to participation. The informed consent process is performed by a member of study staff holding a doctoral degree. Eligible persons are informed of the study purpose, inclusion/exclusion criteria, the possible benefits and risks, their potential involvement and time commitment, and their right to terminate participation at any time without penalty.

Experiment protocol

The study protocol builds on the BO₂ST-I Trial to quantify how tAIH priming enhances the efficacy and potency of training-related functional gains in adults with chronic iSCI (Fig. 1). Participants complete 34 intervention and assessment sessions over 14 weeks at 1 of the 2 clinical sites (Fig. 2). The intervention includes 8 prewash WALK sessions, followed by 16 combinatorial treatment sessions (4 sessions per week for 6 consecutive weeks), and follow-up assessments at 1, 4, and 8 weeks after the final intervention session.

FIG. 2.

Timeline of treatments and assessments. After the initial screening visit (S), each treatment consists of 8 prewash sessions of WALK alone, followed by 16 sessions of tAIH or Placebo prior to WALK_tSTIM. Assessments occur at baseline (BL), at the end of treatment week 2 (T₀), 3 (T₁), 4 (T₂), 5 (T₃), and 6 (T₄), and after 1 week (F₁), 4 weeks (F₄), and 8 weeks (F₈) following the last treatment session. tAIH, therapeutic acute intermittent hypoxia.

tAIH is administered before WALK_tSTIM based on the estimated time course of tAIH-induced BDNF-related plasticity,²⁸ to align these effects with the neuromodulatory mechanisms engaged by gait training and tSTIM. We anticipate that tAIH+WALK_tSTIM will provide greater benefit than WALK alone or Placebo+WALK_tSTIM in improving trained overground walking performance.

Skill-based walking training (WALK)

Participants undertake a total of 24 sessions: 8 during the prewash phase and 16 during the intervention phase, of a skill-based gait training program emphasizing speed, endurance, and balance.²⁰ During the treatment phase, participants receive WALK_tSTIM immediately following (within 30 min) the tAIH or Placebo treatment. A physical or occupational therapist, blinded to the participant’s intervention group assignment, leads each session (see Fig. 3). During high-intensity gait training sessions, heart rate is continuously monitored. Participants will be asked to exercise at moderate to high cardiovascular intensity (70–85% of heart rate reserve), which is known to optimize locomotor recovery and health in individuals with chronic SCI.²⁹ Participants may rest up to 15 min during the practice sessions. During the WALK sessions, the physical therapist trainer will provide physical assistance as needed for safety.

FIG. 3.

Illustration of treatment protocols. (A) A participant receives a tAIH sequence consisting of 15, 1.5-min episodes of breathing low oxygen (10% O₂) with 1-min intervals of breathing room air (21% O₂). (B) We monitor the participant’s heart rate (HR), blood oxygen saturation (SpO₂), and blood pressure (BP) during tAIH treatment. (C) During the tSTIM protocol, participants receive a sequence of 1 ms bursts of electrical stimulation at 30 Hz with 10 kHz carrier frequency that targets the thoracolumbar region (T₁₁-L₂). We set the stimulation amplitude to a subthreshold intensity between 0 and 160 mA before the treatment session. (D) The subthreshold stimulation is active during 45 min of skill-based walking practice (WALK_tSTIM). tAIH, therapeutic acute intermittent hypoxia. Figure adapted from Muter et al. 2023.

Therapeutic acute intermittent hypoxia (tAIH)

Participants undergo 16 sessions over 4 weeks (4 sessions per week) of either tAIH or Placebo, each lasting approximately 37.5 min. Each session consists of 15 episodes of 1.5 min of low oxygen breathing at a fraction of inspired oxygen (F_IO₂) of 0.10 ± 0.02 (∼10% O₂) for tAIH, or room air (F_IO₂ = 0.21 ± 0.02, 21% O₂) for Placebo, separated by 1 min intervals of room air.⁸ tAIH treatments are delivered via an automated air-delivery system,³⁰ with continuous monitoring of blood oxygen saturation and heart rate and blood pressure measurements every five episodes. The U.S. Food and Drug Administration (FDA) has determined the use of the HYP-123 Altitude Generator (Hypoxico, Inc., Gardiner, NY) for this protocol to be nonsignificant risk (NSR) as of March 2025.

Transcutaneous spinal stimulation during WALK training (WALK_tSTIM)

Participants take part in 16 intervention sessions over 4 weeks, 4 sessions per week. During these sessions, up to 60 min of transcutaneous spinal cord stimulation is delivered via a portable spinal stimulator (SCONE; SpineX Inc; Los Angeles, CA) during the WALK_tSTIM.²⁴ The stimulating electrodes are placed at the midline on the trunk superficial to the space between the spinous processes of the T₁₁–T₁₂ and L₁–L₂ vertebrae.³¹ The grounding electrodes are placed over the left and right iliac crests. All electrode sites will be primarily determined via palpation of bony landmarks; however, prior imaging from the participant’s medical records may be used to guide electrode placement in the case that palpation is not possible. The stimulator generates a 30 Hz sequence of biphasic 1 ms bursts with a 10 kHz carrier frequency (Fig. 3). The current amplitude is set to 80% of the motor threshold: the minimum stimulation intensity to elicit an involuntary motor activity in any muscle of either lower extremity.

Outcome measures

To comprehensively evaluate the effects of the intervention and ensure its safety, we employ a set of outcome measures collected at baseline and across multiple time points (T₀, T₁, T₂, T₃, T₄, F₁, F₄, and F₈; see Fig. 2). Data are captured using Research Electronic Data Capture (REDCap) software. These measures are designed to capture changes in functional mobility. We provide a summary of the outcome measures in Table 2.

Table 2.

Functional, Physiological, and Neurocognitive Outcome Measures

Measurement	Data collected	Time collected
Functional assessments
10-Meter Walk Test (10MWT)³²	Time (s)	B, P, I, T, F
6-Minute Walk Test (6MWT)³³	Distance (m)	B, T, F
Timed Up-and-Go Test (TUG)³⁴	Time (s)	B, T, F
Walking Index for SCI II (WISCI II)³⁵	Scalar score (0–20)	B, T, F
SCI Functional Ambulation Inventory (SCI-FAI)³⁶	Scalar score (0–39)	B, T, F
Physiological assessments
Lower Extremity Motor Score (LEMS)³⁷	Scalar score (0–50)	B, T, F
SCI Assessment Tool for Spastic Reflexes (SCATS)³⁸	Scalar score (0–18)	B, T, F
Numeric Pain Rating Scale (NPRS)^39,40	Scalar score (0–10)	B, T, F
Neurogenic Bowel Dysfunction Score (NBDS)⁴¹	Scalar score (0–47)	B, T, F
Neurogenic Bladder Symptom Score (NBSS)⁴²	Scalar score (0–78)	B, T, F
Autonomic Dysreflexia (AD) Incidence Rate	Number of autonomic dysreflexia events
Hypertension Incidence Rate	Blood pressure	B, P, I, T, F
Neurocognitive assessments
California Verbal Learning Test, Third Edition (CVLT3)⁴³	Total recall index score (40–160)	T
Oral Trail Making Test (O-TMT)⁴⁴	Duration (s)	T

If no current/recent record of the ISNCSCI exam is available.

B, baseline; F, follow-up (1, 4, and 8 weeks); I, intervention days; P, prewash days; S, screening day; T, testing days.

Quantifying changes in walking ability

Overground walking ability is our primary outcome measure. We define overground walking ability using three measures: 10MWT (walking speed), TUG (walking initiation and balance), and 6MWT (walking endurance). These measures capture functional walking with high reliability and validity.^45–47 Participants perform two trials each for the 10MWT and TUG at their fastest, safe speed; the average of the two trials is used for analysis. Participants perform one 6MWT at their fastest safe speed. During all walking assessments, participants use their least restrictive assistive device (LRAD). If an LRAD changes during or after the T0 assessment, we will collect assessment data with both the original assistive device and the new device. We provide participants with a minimum of 1 min of rest between tests. Participants perform a single 6MWT at a walking speed sustainable for 6 min. Additional measures of walking function include the Walking Index for Spinal Cord Injury (WISCI) II³⁵ and the Spinal Cord Injury Functional Ambulation Inventory (SCI-FAI).³⁶

Quantifying maladaptive changes

To quantify maladaptive changes that may occur in response to interventions, we measure pain; spasticity; incidence of systemic hypertension and autonomic dysreflexia (AD); and cognitive, bowel, and bladder function.

Pain severity

Pain severity is assessed with an 11-point Numeric Pain Rating Scale (NPRS), which spans from 0 (denoting no pain) to 10 (indicating the most severe pain imaginable). The NPRS has exhibited high test–retest reliability, excellent interpretability, and strong content and construct validity for individuals with SCI.^39,40

Spasticity

Spasticity is evaluated using the Spinal Cord Assessment Tool for Spastic Reflexes (SCATS), which comprises three subscales: lower extremity clonus, flexor spasticity, and extensor spasticity.³⁸

Blood pressure

We define a systemic hypertensive event as a systolic blood pressure (SBP) exceeding 140 mmHg and/or diastolic blood pressure exceeding 90 mmHg.⁴⁸ For each participant, we calculate the hypertension incidence rate, defined as the number of hypertensive events per person-time. We define person-time in units of person-measures (the sum of the total number of blood pressure measurements) taken for each person. Person-measures accounts for the total number of opportunities to detect a hypertensive event and for measurements not made due to dropout or a disqualifying adverse event.⁴⁹

AD incidence rate

AD incidence rate is the number of AD events divided by the total person-days (number of days person remains in the study), accounting for the total number of opportunities to detect AD across days not measured due to dropout or a disqualifying adverse event.⁴⁹ An AD event constitutes a participant having a SBP increase from baseline of 20 mmHg not associated with exercise or SBP > 150 mmHg with complaints of headache, diaphoresis, and/or blurred vision and will be diagnosed by our study team clinicians.

Cognitive function

We assess cognitive ability after the last prewash session and after the last intervention session (T₀ and T₄; Fig. 3).¹¹ The California Verbal Learning Test, Third Edition (CVLT3) Brief Form will quantify verbal learning and memory.⁴³ We also assess executive function with the Oral Trail Making Test (O-TMT).⁴⁴ These tests assess cognitive abilities most likely to be affected by an excessive low oxygen exposure, such as during severe sleep apnea.⁵⁰

Bowel and bladder function

We assess bowel and bladder function using standardized self-reported questionnaires: the Neurogenic Bowel Dysfunction Score (NBDS) version 2.1 and the Neurogenic Bladder Symptom Score (NBSS).^42,51,52 These questionnaires show high test–retest reliability and interpretability.

Quantifying serum hormone levels

We will assess baseline levels of testosterone in our age-stratified experimental arms to determine their potential association with responsiveness to the AIH intervention.

Statistical analysis

This clinical trial will evaluate three prespecified hypotheses using both parametric and nonparametric methods, with statistical significance defined as p < 0.05.

Hypothesis 1: Daily tAIH+WALK_tSTIM improves overground walking ability in persons with chronic iSCI as compared with Placebo+WALK_tSTIM

Changes in 10MWT speed, 6MWT distance, and TUG time compared with post–prewash (T₀) will be analyzed using linear mixed models with fixed effects for intervention (tAIH+WALK_tSTIM vs. Placebo+WALK_tSTIM), age group (younger vs. older), visit, and their interactions, and a random subject effect. We predict greater improvements after 16 sessions of tAIH+WALK_tSTIM than Placebo+WALK_tSTIM, with gains persisting for 8 weeks after training, and we expect similar patterns across the 6MWT and TUG. Exploratory analyses will assess whether treatment effect sizes are associated with baseline and post-treatment testosterone levels using regression models.

To evaluate dose and durability, we will also compare changes in 10MWT, 6MWT, and TUG at 2 versus 4 weeks (8 vs. 16 sessions) of tAIH+WALK_tSTIM, with the expectation that older adults will need more sessions to achieve similar gains, indicating a clinically relevant dose effect.

Hypothesis 2: Daily tAIH enhances the potency of WALK_tSTIM on walking speed to a greater extent in younger (vs. older) persons with chronic iSCI

Subgroup analyses will compare the number of sessions required to reach an MCID in 10MWT speed between interventions (tAIH+WALK_tSTIM vs. Placebo+WALK_tSTIM) and between age groups using survival models for time-to-event data and complementary linear mixed models with session number as the outcome. We predict that younger participants will reach an MCID with fewer sessions than older participants, and that shorter time-to-MCID will be associated with higher baseline testosterone. We also anticipate that, despite slower responses, older participants receiving more sessions will show gains at 4 weeks comparable to younger participants, and that improvements in 10MWT will be paralleled by gains in 6MWT, TUG, SCI-FAI, WISCI II, and Lower Extremity Motor Score (LEMS).

Hypothesis 3: Daily tAIH+WALK_tSTIM does not induce maladaptive changes in persons with chronic SCI

For pain (NPRS), spasticity (SCATS), cognition (CVLT3, O-TMT), bowel function (NBDS), and bladder function (NBSS), we predict no clinically meaningful worsening from baseline with tAIH+WALK_tSTIM relative to Placebo+WALK_tSTIM. These repeated measures will be compared between groups over time using appropriate repeated measures methods (e.g., mixed models or nonparametric equivalents if assumptions are violated), with intervention and time as fixed effects and subject as a random effect. We also predict that tAIH+WALK_tSTIM will not increase the incidence of systemic hypertension or AD.

Safety data will be summarized using frequencies and proportions for categorical variables and means and standard deviations (or medians and interquartile ranges) for continuous variables. Between-group comparisons will use Fisher’s exact test for categorical variables and Student’s t-tests or Wilcoxon rank-sum tests for continuous variables.⁵³ Incidence rates of systemic hypertension and AD will be compared between interventions using relative risk estimates and 95% confidence intervals; a relative risk of 1.0 will indicate no association between intervention and adverse events.

General models and exploratory analyses

All primary models will adjust for prespecified covariates, including study site and key prognostic factors, and will follow an intent-to-treat approach. For participants who do not complete the final evaluation, outcomes will be imputed from the last available assessment, with adverse event-related discontinuations treated conservatively (e.g., imputed as failure for dichotomous outcomes and minimum change for gait speed). These covariate-adjusted models are intended to improve precision and account for any imbalance between groups and sites.⁵⁴ Sensitivity analyses will examine the robustness of findings to model specification, covariate selection, and missing data assumptions, including risks of overfitting, loss of power, and inflated type I error.⁵⁵

Two exploratory analyses are planned. First, the primary models will be refit separately for each site to identify potential site-specific effects. Second, data from BO₂ST-I and BO₂ST-II will be pooled to compare 2-week versus 4-week tAIH pretreatment before WALK_tSTIM using mixed models for repeated measures and to conduct integrated safety analyses given the shared end-points and safety assessments across trials. Mixed models for repeated measures are commonly used to compare the outcome trajectory between groups and within each group.⁵⁶

Blinding integrity

Participants and blinded study team members are informed about potential air mixture conditions (tAIH or Placebo) but remain unaware of the specific treatment assigned. After each session, participants are asked to guess their intervention group and rate their confidence in that guess, with blinding effectiveness to be formally evaluated. In a prior tAIH study using similar procedures, over 75% of participants either guessed incorrectly or were unsure of the treatment they received, supporting the validity of this blinding approach.⁹

Safety precautions

A designated research monitor (medical doctor) oversees the safety of the research and reports observations and findings to the IRB. This includes a review of unanticipated problems involving risks to participants or others and an independent report. See Supplementary Data for further detail on safety measures.

Adverse event reporting

We record adverse events related to each participant from the time of enrollment to the last follow-up assessment visit, including: (1) unintentional loss of balance (i.e., fall to the ground); (2) change in SBP of >140 mmHg and/or diastolic blood pressure exceeding >90 mmHg; (3) AD with SBP > 150 or > 20 mmHg from baseline with complaints of headache, diaphoresis, and/or blurred vision; (4) musculoskeletal injury during/after walking training (i.e., sprain, fracture, etc.); (5) symptoms such as pain, soreness, numbness, or signs of injury (inflammation, blisters, etc.) during or immediately after training or on returning home; (6) hospitalization for any cause; and (7) death attributable to any cause. Serious and/or unexpected adverse events will be immediately reported to the MGB IRB.

Study compensation

Participants are compensated up to $800 and can receive up to $400 in travel reimbursement.

Discussion

This study will examine how age and dose affect responsiveness to tAIH as a primer for skill-based gait training with tSTIM in adults with chronic iSCI. Previous research shows that tAIH, tSTIM, and WALK each enhance walking recovery; however, improvements tend to be modest, slow to develop, and vary across individuals, especially older adults. By using an age-stratified design, extended tAIH dosing, and harmonized methods with the previous BO₂ST-I Trial, BO₂ST-II aims to identify who benefits from tAIH+WALK_tSTIM and how to optimize treatment dosing for better walking recovery.

Age, hormones, and plasticity

Evidence from both pre-clinical and clinical studies suggests that tAIH-induced plasticity depends on serotonin- and BDNF-dependent signaling pathways that are modulated by sex hormones, including testosterone and estrogens. Declines in these hormones with age after SCI, especially beyond 50 years, may reduce the spinal plasticity capacity and explain why older adults have less recovery of functional walking and more variable responses to tAIH. BO₂ST-II directly addresses this gap by enrolling balanced younger (18-49 years) and older (50-80 years) groups, including hormone assessments, and testing whether age influences the extent and durability of tAIH-primed walking improvements.

Dose-response and durability of tAIH priming

Although brief regimens of tAIH can improve lower limb strength⁸ and overground walking when combined with gait training,^9–11 the link between treatment dose and long-term benefits remains unclear. BO₂ST-II extends the tAIH-primed treatment from the 2-week period used in BO₂ST-I to a 4-week course, allowing mixed model and survival analyses across trials to estimate how additional tAIH sessions influence walking gains, the time to achieve clinically meaningful improvement, and the persistence of benefits at follow-up. These dose-finding data, stratified by age, will help determine whether longer tAIH priming is necessary for older adults or if shorter protocols are sufficient for younger individuals with chronic iSCI.

The outcomes of this trial will determine whether 4 weeks of tAIH leads to greater improvements compared with 2 weeks, and whether there is a point where additional sessions provide little or no extra benefit. We will also assess the durability of this intervention by measuring preservation of gains in walking performance after the final tAIH+WALK_tSTIM session. These findings will help guide future trials focused on optimizing tAIH dosing schedules and follow-up periods to achieve meaningful, long-lasting improvements in walking for aging adults with iSCI.

Challenges and opportunities

Conducting randomized clinical trials in people with SCI is challenging, particularly when combining multiple neuromodulation strategies to boost neuroplasticity. This study evaluates a biologically grounded approach that pairs tAIH and tSTIM to engage complementary plasticity mechanisms while delivering intensive, task-specific practice, but feasibility and internal validity remain constrained by real-world enrollment, control, and environmental factors.

Feasibility and validity are threatened by well-known barriers to enrolling and retaining individuals with SCI in rehabilitation trials. This protocol attempts to mitigate these barriers through flexible scheduling, transportation support, rest breaks during sessions, and access to fully accessible temporary housing near the research site, all in line with International Campaign for Cures of Spinal Cord Injury Paralysis (ICCP) recommendations to reduce participant burden and promote adherence.⁵⁷ Despite these measures, limitations in experimental control could still influence the interpretation of treatment effects. The prewash period partially substitutes for a no-treatment control. Still, the study design does not fully capture natural variability in participants’ physical activity outside the study, and the absence of a tSTIM sham condition limits the ability to isolate the independent contribution of each intervention (tAIH, tSTIM, and WALK). Variability in therapist-driven gait progression and unmeasured or partially measured factors such as concomitant serotonergic medications, sleep quality, time-of-day effects, and mild-to-moderate sleep apnea may also confound neuroplasticity and retention, although they will be documented where possible for exploratory and post hoc analyses.

Despite the confounding factors that challenge interpretation of clinical trials in iSCI, this study design represents a crucial step toward understanding the age- and dose-dependent effects of tAIH as a plasticity-enhancing primer for gait training in chronic iSCI. The trial enforces rigor in its design and methods through multicenter, double-blind, randomized procedures; age stratification; guideline-recommended gait outcomes; and harmonized end-points with BO₂ST-I, which collectively support pooled efficacy and safety analyses. Detailed safety monitoring, hormone measurements, and site-specific exploratory models further enhance the mechanistic and translational value of the trial. They will also inform the design of future studies with tighter control over pharmacologic, sleep-related, and environmental factors.

Clinical and translational implications

Given the aging SCI population and the high priority placed on walking recovery, there is a pressing need for scalable, mechanistically informed neuromodulatory interventions that can be tailored across the adult lifespan. Outcomes from BO₂ST-II, together with BO₂ST-I, are expected to (1) define age-appropriate tAIH dosing parameters for use with WALK_tSTIM, (2) clarify whether extended priming improves potency and durability of walking gains, and (3) establish an initial safety and feasibility framework for integrating tAIH into routine gait rehabilitation for chronic iSCI. These results will generate testable hypotheses for future large-scale trials and advance efforts toward personalized, hormone- and age-informed neuromodulation strategies to restore meaningful walking function after SCI.

Authors’ Contributions

J.W.: Methodology, writing—original draft, writing—review and editing, and project administration. N.N. and C.T.: Methodology, writing—original draft, and writing—review and editing. N.P.: Writing—review and editing and project administration. S.P.: Writing—original draft, writing—review and editing, and project administration. H.S. and K.C.: Methodology and writing—review and editing. P.G.: Conceptualization, methodology, funding acquisition, and review/editing final draft. G.S.: Methodology, funding acquisition formal analysis, writing—original draft, and writing—review and editing. G.S.M.: Methodology, writing—review and editing, and funding acquisition. E.F.: Methodology, resources, writing—review and editing, funding acquisition, and supervision. R.D.T.: Conceptualization, methodology, resources, writing—review and editing, supervision, and funding acquisition.

Data Availability

Final trial data are accessible to the study investigators. However, individual data requests should be directed to the principal investigator. The research team plans to disseminate results through published manuscripts and presentations. The authors also intend to make information available according to Spinal Cord Injury Common Data Elements standards, developed through collaboration among the International Spinal Cord Society, the American Spinal Injury Association, and the National Institutes of Health National Institute of Neurological Disorders. At the request of the funding agency’s Program Office, we will submit data for USAMRMC archiving in accordance with the privacy policies of the USAMRMC and institutional review boards at the collaborating sites: Spaulding Rehabilitation Hospital and Brooks Rehabilitation Hospital.

Footnotes

Acknowledgments

The authors thank Angela Link, DPT, NCS, Katherine Schramm, DPT, NCS, Gail Monaghan, PT, ScD, and Angelina Hibberd, OT, OTD, for support with study implementation. They thank Angelina Hibberd, OT, OTD, Lynn McCane, PhD, and Tess Meier, PhD, for assistance with article editing. They thank Dianne Vitkus and Brooke Ellison for their consultation regarding study design. They thank Chloe Slocum, MD, and Kevin O’Connor, MD, for serving as the study physicians.

Author Disclosure Statement

P.G. is the founder and holds shareholder interests in SpineX, Inc.

Funding Information

The authors received financial support for this study from the U.S. Department of Defense (DOD) Spinal Cord Injury Research Program (DOD CDMRP HT94252410708), U.S. Army Medical Research and Materiel Command, Fort Detrick, Maryland 21702-5012. Prior to funding approval, the study protocol underwent peer review. The DOD did not contribute to the study design and does not have ultimate authority in data collection, analyses, or interpretation.

Supplemental Material

Abbreviations Used

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Supplementary Material

Please find the following supplemental material available below.

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A Research Protocol to Study the Dose- and Age-Dependent Effects of Breathing Low Oxygen on Enhancing Transcutaneous Spinal Stimulation-Induced Walking Function for People with Spinal Cord Injury: The BO 2 ST-II Trial

Abstract

Keywords

Introduction

Hypotheses

Methods

Study design

Sample size

Randomization

Study recruitment and enrollment

Experiment protocol

Skill-based walking training (WALK)

Therapeutic acute intermittent hypoxia (tAIH)

Transcutaneous spinal stimulation during WALK training (WALKtSTIM)

Outcome measures

Quantifying changes in walking ability

Quantifying maladaptive changes

Pain severity

Spasticity

Blood pressure

AD incidence rate

Cognitive function

Bowel and bladder function

Quantifying serum hormone levels

Statistical analysis

Hypothesis 1: Daily tAIH+WALKtSTIM improves overground walking ability in persons with chronic iSCI as compared with Placebo+WALKtSTIM

Hypothesis 2: Daily tAIH enhances the potency of WALKtSTIM on walking speed to a greater extent in younger (vs. older) persons with chronic iSCI

Hypothesis 3: Daily tAIH+WALKtSTIM does not induce maladaptive changes in persons with chronic SCI

General models and exploratory analyses

Blinding integrity

Safety precautions

Adverse event reporting

Study compensation

Discussion

Age, hormones, and plasticity

Dose-response and durability of tAIH priming

Challenges and opportunities

Clinical and translational implications

Authors’ Contributions

Data Availability

Footnotes

Acknowledgments

Author Disclosure Statement

Funding Information

Supplemental Material

Abbreviations Used

References

Supplementary Material

Transcutaneous spinal stimulation during WALK training (WALK_tSTIM)

Hypothesis 1: Daily tAIH+WALK_tSTIM improves overground walking ability in persons with chronic iSCI as compared with Placebo+WALK_tSTIM

Hypothesis 2: Daily tAIH enhances the potency of WALK_tSTIM on walking speed to a greater extent in younger (vs. older) persons with chronic iSCI

Hypothesis 3: Daily tAIH+WALK_tSTIM does not induce maladaptive changes in persons with chronic SCI